Summary: Principle Objectives of the Current Research 1. Identification of Bacillus anthracis virulence genes that are induced upon entry into the host through the use of in vivo expression technology (IVET). 2. Identification of Bacillus anthracis virulence genes that are essential for survival following experimental infection, through the use of signature-tagged transposon mutagenesis (STM). 3. Identification of Bacillus anthracis virulence genes that are induced following phagocytosis by monocyte/macrophages, using IVET. 4. Identification of Bacillus anthracis virulence genes that are essential for survival following phagocytosis by monocyte/macrophages, using STM. 5. Development of an animal model of B. anthracis infection. Bacillus anthracis is a Gram (+) rod. The development of 1-2 micron endospores that are resistant to temperature and drying provide the bacterium with a dormant state in order to survive within the soil. Infection is thought to occur by inhalation, abrasion or ingestion. Most commonly, anthrax is diagnosed as a cutaneous infection in those with occupational contact with animals or animal products. Cutaneous anthrax is readily curable using antibiotics and rarely progresses to systemic infection. In contrast, a systemic infection (usually resulting from inhalation of anthrax spores) has a mortality rate approaching 100%. Following inhalation, spores are phagocytosed by alveolar macrophages, triggering germination to the vegetative form. Viable cells are released into the lymphatic system and later into the bloodstream, causing massive bacteraemia of between 107 to 108 cfu/ml. The vegetative form of B. anthracis produces a number of virulence factors including specific toxins. The production of an acute cytokine response contributes to hypotension, septic shock and death. Specific genes are induced in response to environmental conditions that signal entry into host tissues. We are currently developing tissue culture and animal models of B. anthracis infection to facilitate the identification of genes involved in the early steps of B. anthracis infection by the pulmonary route. We are also developing genetic tools to allow for the screening and selection of mutants defective in these steps. The IVET "promoter trap" strategy provides a method to identify genes that are induced upon host entry or when B. anthracis is phagocytosed by a macrophage. In contrast, STM provides a method to identify genes that are necessary for the survival of the pathogen during the infective process. The rationale for the use of both the IVET and STM methods is based on observations that these two systems are complementary, and have been shown to yield different results. We anticipate undertaking our initial screens and selections in the first half of the coming year.